This study aimed to investigate the phytochemical content and biological activity and of Atriplex farinosa by studying its effects on male fertility. Five compounds were isolated from this plant (Quercetin-4/-methoxy-7-gluco-rhamnoside, Kaempferol -4/- methoxy - rutinoside, Quercetin -6, 4/- dimethoxy - 3 - gluco-rhamnoside, Scopolin and Scopoletin), also the results revealed that A. farinosa showed a significant activity on male fertility by reducing the studied parameters such as weight of reproductive organs, percent of unstained live sperms, total sperms abnormality and testosterone.
Keywords: Atriplex farinosa, coumarins, kaempferol, kaempferol-7-rhamnoside, kaempferol-3,7-dirhamnoside, male fertility, quercetin, phytochemical studies,
Introduction.
Atriplex farinosa (Family Chenopodiaceae) is a tall, robust shrub of yellow white appearance with large, naked panicles, but leaf base cordate with long, obtuse auricles, fruit bracts entire, longer than broad, spathulate, acute (Täckholm, 1974)1. Among the halophyte flora, species belonging to the genus Atriplex of special interest because of their high biomass production associated with a deep root system able to cope with the poor structure, these species also naturally produce high amounts of oxalic acid, which may assume positive functions in tolerance mechanisms to heavy metal stress (2-3). Among Chenopodiaceae the genus Atriplex is probably, the most studied, probably because many species are used for rehabilitation of saline soils4. These plants could be promising, since Atriplex species have special bladders in the leaves that act as salt sinks for the removal of the excess of salt5. Plants in the genus Atriplex (Chenopodiaceae) have been proposed as possible candidates for phytoremediation of Se, these plants could improve grower participation in phytoremediation 6.
Earlier works suggested the presence the naringin, naringenin 7-O glucoside, isorhamnetin 3-O-rhamnosyl (1-6) glucopyranoside and isorhamnetin 7-Oglucopyranoside in A. farinosa (Al jabber 19917).
Atriplex halimus is rich in fiber, protein and numerous trace minerals including chromium. The study of Clinton (1998)8 indicated that Atriplex halimus improved blood glucose regulation and glucose tolerance in diabetes mellitus. Dosage used in this study was 3g per day. A significant increase in serum ALT in camels fed on Atriplex halimus plus 75 % barley of maintenance energy requirement and 25% olive cake for six months, while serum creatinine did not change in two groups of camels fed on Atriplex halimus plus 100 % barley and 75 % barley plus 25 % olive cake for 6 months. El-Bashary (2000) 9
Feeding goats on Atriplex halimus and Acacia saligna, led to decreasing total serum proteins and serum albumin compared with control group. There was no significant increase in serum globulin in both Atriplex and Acacia groups, also there was a gradual significant increase in serum ALT in sheep fed on Atriplex halimus, and serum AST also increased, in addition there was an increase in serum urea and serum creatinine in sheep fed on Atriplex halimus in comparison with control group (Ibrahim, 2001)10.
The effect of sublethal concentrations of Atriplex halimus L. extract on mortality rate, longevity, egg production of Biomphalaria alexandrina and hatchability of their eggs was done. This extract caused reduction in all the previously mentioned parameters, this decreased was correlate with the increase in plant concentrations Tantawy (2002)11.
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea and serum creatinine were increased in sheep fed on Atriplex lentiformis for 45 days, also reduction in live body weight occurred (Sabry et al., 2003)12. Various species of Atriplex have medicinal values, e.g. Atriplex semibacata has been used as antifungal and Atriplex vestita in the treatment of bronchitis. Atriplex hortensis has been regarded as a source of vitamin A, (Siddiqui et al., 1994)13.
In traditional medicine, a cocktail of minerals in A. halimus is used to benefit glycaemic control in diabetic patients 14. Like other halophytes, it can combat internal parasites in veterinary use 15. A. halimus produce the polyphenols and other bioactive substances potentially useful for medicinal properties and as natural food preservation. The distribution of these molecules was unequal in different parts of plant. The leaves exhibited the higher phenolic content in comparison with the stems. However, the flavonoids in ethyl acetate and butanolic fractions possess potential antioxidant activity which explains the relation structure-activity. (Nabila Benhammou et al 2009)16.
Atriplex confertifolia has significant bioactivity against human breast cancer cell lines, the bioactivity of A. confertifolia extracts on these cells lines was compared to an FDA-approved cancer drug (Onxol) and an industry-standard leukocyte control cell line. Active portions of the extracts were found primarily in the polar fractions of the plant. A dose-response curve of the extracts displayed significant cell death similar to Onxol (Capua et al., 2010)17.
Material and Methods.
Plant Material. Atriplex farinosa Forssk. (Collected in the wild from the southeastern corner of Egypt), during autumn season 2007. The collected plant, identified by Prof. Dr. Ahmed Morsy Ahmed, Plant Ecophysiology, Desert Research Center, Cairo, Egypt, the plant were dried under shade and then grinded to fine powder. The dried powder 1k g was extracted with 70% aqueous eethanol by percolation in the solvent with occasional shaking for 48 hours and this was repeated three times. The ethanolic extract was combined and concentrated under reduced pressure to give 75g.
The ethanolic extract of Atriplex farinosa (25 g) was separated in to fractions on a silica gel (450 g, mesh 60-120) column (5 cm in diameter x 130 cm in length) and eluted with ethyl acetate (fractions 1-8), and ethyl acetate:methanol (95:5, v:v) (fractions 9- 21), and ethyl acetate:methanol (80:20, v:v) (fractions 22-37). Fractions (1-8) were applied to a silica gel (100 g of mesh 60-120) column (3 cm in diameter x 70 cm in length) eluted with chloroform, from which compounds 4 and 5 were isolated. Fractions 22-37 (5 g) were applied to a second silica gel (150 g of mesh 60-120) column (3 cm in diameter x 90 cm in length) and eluted with ethyl acetate:methanol:water (70:5:4, v:v:v) and then with a ethyl acetate:methanol:water (30:5:4, v:v:v), from this column compounds 1, 2 and 3 were isolated.
Acid Hydrolysis.
From the recrystalllized compounds, 2 mg of each were dissolved in 2 mL of methanol:water (1:1, v:v), mixed with 1 mL of 2N HCl, and refluxed at 60 oC for 3 h. The reaction mixture was subsequently extracted with ethyl acetate to give a glycone moiety and the neutralized aqueous part afforded sugars moiety (Stahl 1969)18
Apparatus:
UV spectra of the isolated compounds were measured on Shimadzu 1201 spectrophotometer. Mass spectral data were done using Esquire-LC_00142 mass spectrometer. 1H and 13C NMR spectra, using external electronic referencing through the deuterium resonance frequency of the solvent, were determined at 400 and 100 MHz respectively with Avance NMR spectrometer fitted with an auto-tune 5mm X/H probe. 1H-13C correlations were established by using HMQC and HMBC pulse sequences respectively. 1H-1H correlations were determined by double quantum filtered COSY.
Toxicological Studies:
LD50 values of the tested extracts were determined by a known method (Finney 1964)19. Rats were divided into groups of 6 animals each. Preliminary experiments were done to determine the minimal dose that killed all rats and the maximal dose that failed to kill any animal. Several doses at equal intervals were administrated orally to a group of 6 rats. Animals were kept under observation for 24 hr during which symptoms of toxicity and rate of mortality in each group were recorded from which the LD50 therapeutic dose was calculated.
Determination of Prolactin, testosterone, FSH and Luteinizing Hormone were determined according to the method described by Tietz (1995)20, Chen et al., (1991)21 and Uotila et al., (1981)22.
Results and discussion:
1- Isolated compounds:
Compound (1) was obtained as yellow crystals (10 mg), (m.p. 230 oC) soluble in methanol. It gave positive result with Molisch's test The EI-mass spectrum of compound 1 revealed peaks at (M-1) 648 and other important peaks at M/e 331(100%). UV: λ max (MeOH): (nm) 254, 354, (NaOMe) 272, 401, 406, (AlCl3) 269, 381, (AlCl3/ HCl) 268, 379, (NaOAc) 273, 321,369, (NaOAc/H3BO3) 254,266,355. 1HNMR(MeOD), δ: δ 7.62 integrated for one proton J = 8.5 Hz for 2', δ 7.47 (1H, dd, J = 8.5, J = 2.5 Hz, H-6' ), δ 6.87 (1H, d, J = 8.5 Hz, H-5' ), δ 6.22 (1H, d, J = 2.5 Hz, H-8 ), δ 6.07 (1H, d, J = 2.5 Hz ,H-6), δ 5.1 (1H, d, J = 2 Hz, H1'' rhamnose) , δ 4.52 (H , d, J = 2 Hz, H1''' glucose), δ 3.9 (3H , OCH3 ), δ 3.20-3.7 ( m, remaining sugar protons) and δ 1.17(3H, d, J = 6 Hz, CH3 rhamnose). 13C NMR (MeOD):177.4 (C-4), 164 (C-7), 162.69 (C-5), 157.01 (C-2), 156.90 (C-9), 149.93 (C-4'), 147.39 (C-3').133.49 (C-3),122.76 (C-6'), 121.54 (C-1'), 113.71 (C-2'), 115.76 (C-5') 104.27(C-10). 101.43 (C-1'''), 101.77 (C-1''') 99.36 (C-6), 94.38 (C-8), the remaining sugar carbons appeared at 63.6-76.89, 56.29(-OCH3), 18.24(C-6'''). This compound was identified as quercetin-4/-methoxy-7-gluco-rhamnoside.
Compound (2) was obtained as yellow crystals (20 mg), (m.p.228-33oC) soluble in methanol. UV: λmax (MeOH): (nm) 271, 323, 336, (NaOMe) 275, 400, (AlCl3) 272, 353, (AlCl3/ HCl) 274, 360, (NaOAc) 272, 352 (NaOAc/H3BO3) 272,353. 1HNMR (MeOD), δ 7.99 integrated for two protons J = 8.5 Hz for 2'and 6', δ 6.89 (2H, d, J = 8.5, H-3'-5' ), δ 6.8 ( 1H, d, J = 2.5 Hz, H-8 ), δ 6.51 ( 1H, d, J = 2.5 Hz, H-6 ), δ 5.32 (1H, d, J = 2 Hz, H1'' rhamnose) , δ 4.37 ( 1H , d, J = 2 Hz, H1''' glucose), δ 3.70 (3H , OCH3 ), δ 3.0-3.75 (m, remaining sugar protons) and δ 0.97 ( 3H, d, J = 6 Hz, CH3 rhamnose). 13C NMR (MeOD): 178.03 (C-4), 160 (C-7), 58.52 (C-5), 157.34 (C-2), 152.30 (C-9), 149.93 (C-4'), 147.39 (C-3').133.37(C-3), 131.38 (C-6', C-2'), 121.54 (C-1'), 115.76 (C-5') 104.27(C-10). 101.43 (C-1''), 101.72 (C-1''') 99.36 (C-6), 94.38 (C-8), the remaining sugar carbons appeared at 63.6-76.89, 56.29(-OCH3), 18.24(C-6'''). This compound was identified as kaempferol-4/-methoxy-rutinoside.
Compound (3) was obtained as yellow crystals (35 mg), (m.p 232 oC) soluble in methanol. It gave positive result with Molisch's test, UV: λmax ( MeOH): (nm ) 255, 348, 350, (NaOMe) 272, 334, 385, ( AlCl3) 270, 380, ( AlCl3/ HCl) 269, 380, (NaOAc) 255, 361, (NaOAc/H3BO3) 258, 362. 1HNMR (MeOD), δ 7.89(1H, dd, J = 8.5 Hz, J =2.5 Hz, H- 2'), 7.62 (1H, dd, J = 8.5 Hz, J =2.5 Hz, H-6'), 6.91 (1H, d, J = 8.5 Hz, H-5'), 6.47 (1H, s, H-8 ). 5.21 (1H, d, J = 2 Hz, H1'' rhamnose) , 4.52 (1H , d, J = 2, H1'''), 3.95, 3.87 sugar protons, 3.28-3.36 (m, remaining sugar protons) and 1.1 ( 3H, d, J = 6, CH3 rhamnose. 13C NMR (MeOD): 179.2 (C-4), 158.64 (C-7), 150.81 ( C-5), 149.20 (C-2), 148.30 (C-9), 147.38 ( C-4'), 135.06 (C-3'), 132.1 (C-3), 123.95 (C-6'), 123.00 (C-1'), 116.07 ( C-2'), 114.46 ( C-5'), 104.51(C-10), 102.48 (C-1''), 101.90 (C-1''') 98.8 (C-6), 93.7 (C-8), 60.88 and 56.37 (two -OCH3 groups), The remaining sugar carbons appeared at 63.6-76.9, 18.24 C-6''. Mass spectrum (M-1) 678 and another important peak at M/e 331(100%), also from DQF cosy HMQC and HMBC data this compound was identified as quercetin -6, 4/- dimethoxy - 3 - gluco-rhamnoside.
Compound 4: (27 mg) white crystals, m.p. 203-204 oC. λmax (MeOH): (nm) 229,252,261, 294, 346., (NaOAc) 244,277,390. 1H NMR (DMSO-d6) δ 7.92 (1H, d, J =9 , H-4 ) , δ 7.2(1H, s,H-5 ), δ 6.75 ( 1H, s, H-8), δ 6.2 (1H , d, J =9, H-3) and δ 3.8 (3H, s, OCH3)., This compound was identified as Scopoletin (23).
Compound 5: (25 mg) white crystals, m.p. 126 -128 oC UV: λmax (MeOH): (nm) 276, 349, (NaOAc) 259, 390. 1HNMR (DMSO-d6) δ 7.93 (1H, d, J = 9.5, H-4), δ 7.3 (1H, d, J = 8.4, H-5), δ 7.1 (1H, dd, J = 8.4, 2.2, H-8), δ 6.3 (1H, d, J = 9.5, H-3), and 3.8 (3H, s, OCH3). δ 5.1 (1H, d , J = 9, H-1' glucose) and δ 3-3.8 sugar protons. 13C NMR (DMSO-d6) 160.2 (C-2), 1121 (C-3), 145.8 (C-4), 109.5 (C-5), 113.2 (C-6), 149.8 (C-7), 102.8 (C-8) , 153.6 (C-9), 112.3 (C-10), 100.6 (C-1'), 73.3 (C-2' ), 76.7 (C-3' ), 69.4 (C-4' ), 77 (C-5' ), 61 (C-6' ) and 55.9 (OCH3). This compound was identified as scopolin
Compound
R1
R2
R3
R4
R5
1
rha-glu.
H
OH
OCH3
OH
2
OH
H
rutioside
OCH3
H
3
OH
OCH3
rha-glu.
OCH3
OH
Compound
R
R1
4
OH
OCH3
5
Glucose
OCH3
Fig. (1): Isolated compounds from Atriplex farinosa.
2-Pharmacological Studies:
2.1.Toxicological Studies
•Acute toxicity and median lethal dose (LD50) test:
Atriplex farinosa in oral doses up to 5000 mg/kg b.wt. did not produce any demonstrable acute toxic effects or deaths in all groups of rats.
2.2. Effect on male fertility:
Oral administration of the ethanolic extract of Atriplex farinsa for six weeks showed a significant effects on weight of reproductive organs including reduction in the weight of testes, seminal vesicles and ventral prostate (table 1). Semen characters also showed a significant reduction, this appeared in sperm count, sperm mobility and unstained live sperm (table 2). Atriplex farinosa extract showed a lowering in the testosterone level and FSH, it showed an increase in Prolactin level compare to control group (table 3).
These results in slightly in the same trend with that achieved by Tantawy (2002) in which mortality rate, longevity, egg production of Biomphalaria alexandrina and hatchability of their eggs were reduced by feeding Atriplex halimus.
It can be concluded that A.farinosa can be used for the reduction of fertility of unwanted animals and pits.
Table (2): Effect of oral administration of ethanolic extract of Atriplex farinosa for 6 successive weeks on weights of sexual organs of male rats, (n=6).
Groups
Initial b.wt (g)
Final b.wt (g)
Weight of reproductive organs (g/100 g b.wt)
Testes (Pair)
Seminal vesicles
Ventral prostate
Control
225.8±6.83
238.2±6.57
1.65±0.11
0.53±0.03
0.48±0.01
A. farinosa
224.2±6.28
235.3±6.49
1.16±0.08**
0.41±0.02**
0.40±0.02**
Table (3): Effect of oral administration of ethanolic extract of Atriplex farinosa for 6 successive weeks on semen characteristics of male rats, (n=6).
Groups
Sperm count (X 106/mL)
Sperm motility (%)
Unstained (live) sperms (%)
Total sperm abnormality (%)
Control
62.78±3.55
92.70±3.68
92.52±3.88
3.11±0.26
A. farinosa
52.65±2.61*
86.43±3.72
81.15±3.26*
4.24±0.31*
Table (4): Effect of oral administration of ethanolic extract of Atriplex farinosa for 6 successive weeks on plasma levels of reproductive hormones of male rats, (n=6).
Groups
Testosterone (ng mL-1)
Prolactin
(ng mL-1)
FSH
(mIU mL-1)
LH
(mIU mL-1)
Control
4.42±0.35
0.68±0.05
7.38±0.25
0.59±0.02
Atriplex farinosa
2.91±0.24**
1.15±0.11**
7.07±0.29
0.62±0.04
Acknowledgements
The authors are thankful to Dr for his help and support during
